Abstract: RNA localization is an important mechanism for regulating the spatial distribution of proteins inside the cell. Defects in mRNA localization have been shown in different organisms to disrupt the formation of a normal body plan and to inhibit the function of the central nervous system. In humans, the loss of a single mRNA that is localized to dendrites leads to Fragile X syndrome, which is the most common inherited form of mental retardation. There is mounting evidence that RNA localization plays a large role in the spatial cell biology of many organisms, but studies are often limited to static snapshots of this intrinsically dynamic cellular process. Existing strategies for fluorescent labeling of RNAs have several drawbacks when applied to live cells, a major limitation being high background fluorescence. Thus, there remains a critical need for technical advances in the field of live cell imaging of RNAs. In this proposal, we present an innovative strategy for tagging RNAs with fluorescent small molecule probes. This method should have several advantages for fluorescence imaging applications, including specific targeting, low background, and interchangeability of the small molecule probes. We have developed a synthetic strategy to access two classes of fluorescent ligand probes. The cell permeability and binding affinity of these new compounds to the corresponding RNA tags will be tested. We will then apply this chemical tagging system to study the localization of mRNAs encoding protein paralogs in yeast. We also will develop the system for use in other organisms through collaborative projects. Finally, we will explore other functions for the chemical handle appended on the tagged RNA, including affinity purification and selective inactivation of the RNA and its associated protein partners. This new fluorescent labeling strategy, which involves chemically tagging RNAs in live cells, is expected to provide a powerful tool for basic research into RNA localization. Public Health Relevance: The asymmetric distribution of RNAs inside cells is required for the function of specialized cells like neurons and for the development of a normal body plan. In order to understand how and why certain RNAs are localized, we propose to develop a technique to chemically tag RNAs for live cell imaging. Furthermore, the utility of our tagging strategy for other applications, including isolating RNA-protein complexes and inactivating RNAs, will be investigated.